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EC number: 948-055-8 | CAS number: -
- Life Cycle description
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 April 2018 - 30 April 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 31 May 2008
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Extract obtained from the shell of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol
- EC Number:
- 948-055-8
- Molecular formula:
- not applicable as it is a UVCB
- IUPAC Name:
- Extract obtained from the shell of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol
- Test material form:
- liquid: viscous
- Details on test material:
- - Physical appearance: dark brown to black viscous liquid
- Storage conditions: in refrigerator (2-8°C) protected from light
Constituent 1
Method
- Target gene:
- Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal enzymes (S9), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).
- Test concentrations with justification for top dose:
- Dose range finding test (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of the first experiment)
First experiment (TA1535, TA 1537 and TA98, with and without S9): 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (all strains, with and without S9): 52, 164, 512, 1600 and 5000 μg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: The test item formed a clear brown solution in Milli-Q water. The vehicle was according to OECD guideline 471.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191; 2-aminoanthracene
- Remarks:
- For more details see 'any other information on materials and methods'
- Details on test system and experimental conditions:
- Two individual experiments were performed: the first experiment was a direct plate assay and the second experiment was a pre-incubation assay. A dose-range finding study was performed and reported as part of the first experiment.
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 +/- 4 hours (for both experiments)
- Pre-incubation period: 30 +/- 2 minutes (for the second experiment only)
NUMBER OF REPLICATIONS: 3
PLATE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h.
NUMBER OF CELLS EVALUATED: 10^9 cells/mL
CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies
COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope. - Evaluation criteria:
- A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in both experiments, no precipitation was observed at the start or at the end of the incubation period in any of the strains and at any of the doses tested.
CYTOTOXICITY:
- Experiment 1: No cytotoxicity observed in any of the tester strains.
- Experiment 2: No cytotoxicity observed in any of the tester strains. Except in tester strain TA1537 in the absence of S9-mix where fluctuations in the number of revertant colonies below the laboratory historical control data range were observed at the dose level of 5000 µg/plate. However since the reduction in the mean number of revertant colonies was only minor when compared against relevant historical control data and no toxicity was observed in any of the other tester strains in both experiments, this reduction is caused by incidental fluctuations in the number of revertant colonies and is considered as not biologically relevant.
MUTAGENICITY: In both experiments, in all strains and at all doses tested, no biologically relevant increase in the number of revertants was observed upon treatment with the test item, with and without S9.
HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Any other information on results incl. tables
Table 2 Historical data for the solvent control
|
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
|||||
S9-mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
Range |
3 – 29 |
3 – 27 |
3 – 20 |
3 – 23 |
8 - 41 |
8 – 55 |
61 – 176 |
60 - 160 |
10 – 59 |
9 - 67 |
Mean |
10 |
11 |
6 |
6 |
16 |
22 |
110 |
106 |
26 |
33 |
SD |
3 |
4 |
2 |
3 |
5 |
7 |
17 |
20 |
6 |
8 |
n |
2458 |
2426 |
2402 |
2352 |
2416 |
2458 |
2473 |
2398 |
2237 |
2217 |
Table 3 Historical data for the positive control
|
TA1535 |
TA1537 |
TA98 |
|||
S9-mix |
- |
+ |
- |
+ |
- |
+ |
Range |
128 – 1530 |
73 – 1206 |
58 – 1407 |
54 – 1051 |
365 – 1978 |
250 – 1977 |
Mean |
901 |
239 |
817 |
340 |
1355 |
903 |
SD |
174 |
115 |
354 |
160 |
230 |
357 |
n |
2400 |
2296 |
2051 |
2337 |
2357 |
2367 |
|
TA100 |
WP2uvrA |
||
S9-mix |
- |
+ |
- |
+ |
Range |
439 – 1993 |
408 - 2379 |
93 – 1958 |
111 - 1359 |
Mean |
905 |
1249 |
1059 |
444 |
SD |
163 |
371 |
506 |
144 |
n |
2402 |
2354 |
2153 |
2232 |
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Oct 2015 and Oct 2017.
Applicant's summary and conclusion
- Conclusions:
- The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that Cocoa Shell Extract (Pot) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
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